Abstract

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.

Plk1 is activated during ciliary disassembly but has limited influence on disassembly dynamics.

A Immuno blot analysis of whole cell lysates prepared from hTERT-RPE1 grown under starved, ciliated conditions (0h) or at the times indicated following serum treatment to induce ciliary resorption. Ph-Plk1T210 represents activated Plk1; B, C Quantification of the expression of (B) Ph-Plk1T210 or (C) total Plk1 normalized to β-actin and relativized to the expression level without serum induction. Results from three independent experiments were calculated. * P<0.05, ***P≤0.005. Error bars represent SE. D Upper panel: Immunofluorescence of starved ciliated hTERT-RPE1 cells treated with the Plk1-inhibitor BI 2536 or with DMSO for 3 hours, then maintained in serum-free medium, or induced for ciliary disassembly with 10% serum-containing medium. Lower panel: Immunofluorescence of serum-starved, ciliated hTERT-RPE1 cells treated with siRNA to Plk1 or with scrambled (scr) control siRNA after 0, 2 and 24 hours of serum addition. Image shows acetylated α-tubulin (green), γ-tubulin (red) and DNA (blue). The scale bar represents 10 µm. E - H Quantification of three independent repetitions of experiment presented in D. E and G Quantification of the percentage of ciliated cells, with an average of 250 cells counted for each condition. F and H Quantification of cilia length, with an average of 50 cilia measured for each condition. * P<0.05, **P<0.01. ***P<0.001. Error bars represent SE.